The Pachysolen CUG-tRNA is an Ala-tRNA. (A) Secondary structures of the Pachysolen tannophilus ${\text{tRNA}}_{\mathrm{CAG}}^{\mathrm{Ala}}$, the Candida albicans ${\text{tRNA}}_{\mathrm{CAG}}^{\mathrm{Ser}}$, and the Yarrowia lipolytica ${\text{tRNA}}_{\mathrm{CAG}}^{\mathrm{Leu}}$. The consensus Saccharomycetales ${\text{tRNA}}_{\mathrm{HGC}}^{\mathrm{Ala}}$ (based on 752 sequences), ${\text{tRNA}}_{\mathrm{HGA}}^{\mathrm{Ser}}$ (748 sequences), and ${\text{tRNA}}_{\mathrm{NAG}}^{\mathrm{Leu}}$ (300 sequences) are shown for comparison. The discriminator base “N73” is denoted by the corresponding nucleotide number of the respective tRNA gene. It is obvious that the Pachysolen ${\text{tRNA}}_{\mathrm{CAG}}^{\mathrm{Ala}}$ and the Candida ${\text{tRNA}}_{\mathrm{CAG}}^{\mathrm{Ser}}$ share more consensus elements with the alanine and serine tRNAs, respectively, than with the leucine tRNAs. tRNA identity determinants are indicated by red boxes and dashed lines. (B) Unrooted phylogenetic network of 172 tRNA sequences generated using the neighbor-net method as implemented in SplitsTree v4.1.3.1. Methionine, isoleucine, arginine, and threonine tRNAs were included as outgroup. ${\text{tRNA}}_{\mathrm{CAG}}^{\mathrm{Leu}}\mathrm{s}$, ${\text{tRNA}}_{\mathrm{CAG}}^{\mathrm{Ser}}\mathrm{s}$, and the Pachysolen ${\text{tRNA}}_{\mathrm{CAG}}^{\mathrm{Ala}}$ are highlighted in dark green, blue, and red, respectively.